RESUMEN
Hyperferritinemia is a frequent finding in several conditions, both genetic and acquired. We previously studied eleven healthy subjects from eight different families presenting with unexplained hyperferritinemia. Their findings suggested the existence of an autosomal-recessive disorder. We carried out whole-exome sequencing to detect the genetic cause of hyperferritinemia. Immunohistochemistry and flow cytometry assays were performed on liver biopsies and monocyte-macrophages to confirm the pathogenic role of the identified candidate variants. Through a combined approach of whole-exome sequencing and homozygosity mapping, we found bi-allelic STAB1 variants in ten subjects from seven families. STAB1 encodes the multifunctional scavenger receptor stabilin-1. Immunohistochemistry and flow cytometry analyses showed absent or markedly reduced stabilin-1 in liver samples, monocytes, and monocyte-derived macrophages. Our findings show a strong association between otherwise unexplained hyperferritinemia and bi-allelic STAB1 mutations suggesting the existence of another genetic cause of hyperferritinemia without iron overload and an unexpected function of stabilin-1 in ferritin metabolism.
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Hiperferritinemia , Sobrecarga de Hierro , Humanos , Sobrecarga de Hierro/genética , Sobrecarga de Hierro/diagnóstico , Ferritinas/genética , Macrófagos , AlelosRESUMEN
OBJECTIVE: The MC1-R (melanocortin 1 receptor) is expressed by monocytes and macrophages where it mediates anti-inflammatory actions. MC1-R also protects against macrophage foam cell formation primarily by promoting cholesterol efflux through the ABCA1 (ATP-binding cassette transporter subfamily A member 1) and ABCG1 (ATP-binding cassette transporter subfamily G member 1). In this study, we aimed to investigate whether global deficiency in MC1-R signaling affects the development of atherosclerosis. APPROACH AND RESULTS: Apoe-/- (apolipoprotein E deficient) mice were crossed with recessive yellow (Mc1re/e) mice carrying dysfunctional MC1-R and fed a high-fat diet to induce atherosclerosis. Apoe-/- Mc1re/e mice developed significantly larger atherosclerotic lesions in the aortic sinus and in the whole aorta compared with Apoe-/- controls. In terms of plaque composition, MC1-R deficiency was associated with less collagen and smooth muscle cells and increased necrotic core, indicative of more vulnerable lesions. These changes were accompanied by reduced Abca1 and Abcg1 expression in the aorta. Furthermore, Apoe-/- Mc1re/e mice showed a defect in bile acid metabolism that aggravated high-fat diet-induced hypercholesterolemia and hepatic lipid accumulation. Flow cytometric analysis of leukocyte profile revealed that dysfunctional MC1-R enhanced arterial accumulation of classical Ly6Chigh monocytes and macrophages, effects that were evident in mice fed a normal chow diet but not under high-fat diet conditions. In support of enhanced arterial recruitment of Ly6Chigh monocytes, these cells had increased expression of L-selectin and P-selectin glycoprotein ligand 1. CONCLUSIONS: The present study highlights the importance of MC1-R in the development of atherosclerosis. Deficiency in MC1-R signaling exacerbates atherosclerosis by disturbing cholesterol handling and by increasing arterial monocyte accumulation.
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Aorta/metabolismo , Enfermedades de la Aorta/metabolismo , Aterosclerosis/metabolismo , Ratones Noqueados para ApoE , Monocitos/metabolismo , Placa Aterosclerótica , Receptor de Melanocortina Tipo 1/deficiencia , Transportador 1 de Casete de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Animales , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Aterosclerosis/genética , Aterosclerosis/patología , Células Cultivadas , Colesterol/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Hígado Graso/genética , Hígado Graso/metabolismo , Hígado Graso/patología , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones Endogámicos C57BL , Monocitos/patología , Receptor de Melanocortina Tipo 1/genéticaRESUMEN
PURPOSE: Macrophages are critical in driving an immunosuppressive tumor microenvironment that counteracts the efficacy of T-cell-targeting therapies. Thus, agents able to reprogram macrophages toward a proinflammatory state hold promise as novel immunotherapies for solid cancers. Inhibition of the macrophage scavenger receptor Clever-1 has shown benefit in inducing CD8+ T-cell-mediated antitumor responses in mouse models of cancer, which supports the clinical development of Clever-1-targeting antibodies for cancer treatment. PATIENTS AND METHODS: In this study, we analyzed the mode of action of a humanized IgG4 anti-Clever-1 antibody, FP-1305 (bexmarilimab), both in vitro and in patients with heavily pretreated metastatic cancer (n = 30) participating in part 1 (dose-finding) of a phase I/II open-label trial (NCT03733990). We studied the Clever-1 interactome in primary human macrophages in antibody pull-down assays and utilized mass cytometry, RNA sequencing, and cytokine profiling to evaluate FP-1305-induced systemic immune activation in patients with cancer. RESULTS: Our pull-down assays and functional studies indicated that FP-1305 impaired multiprotein vacuolar ATPase-mediated endosomal acidification and improved the ability of macrophages to activate CD8+ T-cells. In patients with cancer, FP-1305 administration led to suppression of nuclear lipid signaling pathways and a proinflammatory phenotypic switch in blood monocytes. These effects were accompanied by a significant increase and activation of peripheral T-cells with indications of antitumor responses in some patients. CONCLUSIONS: Our results reveal a nonredundant role played by the receptor Clever-1 in suppressing adaptive immune cells in humans. We provide evidence that targeting macrophage scavenging activity can promote an immune switch, potentially leading to intratumoral proinflammatory responses in patients with metastatic cancer.
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Moléculas de Adhesión Celular Neuronal , Activación de Linfocitos , Neoplasias , Receptores Mensajeros de Linfocitos , Humanos , Linfocitos T CD8-positivos/inmunología , Moléculas de Adhesión Celular Neuronal/antagonistas & inhibidores , Regulación hacia Abajo , Activación de Linfocitos/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Receptores Mensajeros de Linfocitos/antagonistas & inhibidoresRESUMEN
PURPOSE: As foremost regulators of cancer-related inflammation and immunotherapeutic resistance, tumor-associated macrophages have garnered major interest as immunotherapeutic drug targets. However, depletory strategies have yielded little benefit in clinical studies to date. An alternative approach is to exploit macrophage plasticity and "reeducate" tumorigenic macrophages toward an immunostimulatory phenotype to activate the host's antitumor immunity.Experimental Design: We investigated the role of the macrophage scavenger receptor common lymphatic endothelial and vascular endothelial receptor-1 (Clever-1) on tumor growth in multiple mouse cancer models with inflammatory and noninflammatory characteristics by using conditional knockouts, bone marrow chimeras, and cell depletion experiments. In addition, the efficacy of immunotherapeutic Clever-1 blockade as monotherapy or in combination with anti-PD-1 was tested. RESULTS: Genetic deficiency of macrophage Clever-1 markedly impaired solid tumor growth. This effect was mediated by macrophages that became immunostimulatory in the absence of Clever-1, skewing the suppressive tumor microenvironment toward inflammation and activating endogenous antitumor CD8+ T cells. Comparable effects were achieved with immunotherapeutic blockade of Clever-1. Notably, these effects were similar to those achieved by PD-1 checkpoint inhibition. Moreover, combining anti-Clever-1 with anti-PD-1 provided synergistic benefit in aggressive, nonresponsive tumors. CONCLUSIONS: These findings demonstrate the importance of macrophages in mediating antitumor immune responses and support the clinical evaluation of immunotherapeutic Clever-1 blockade as a novel cancer treatment strategy.See related commentary by Mantovani and Bonecchi, p. 3202.
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Linfocitos T CD8-positivos/inmunología , Neoplasias , Animales , Inmunoterapia , Macrófagos/efectos de los fármacos , Ratones , Microambiente Tumoral/efectos de los fármacosRESUMEN
Clever-1, encoded by the Stab1 gene, is a scavenger and leukocyte trafficking receptor expressed by subsets of vascular and lymphatic endothelial cells and immunosuppressive macrophages. Monocyte Clever-1 also modulates T cell activation. However, nothing is known about the possible links between B cell function and Clever-1. Here, we found that Stab1 knockout mice (Stab1-/-) lacking the Clever-1 protein from all cells present with abnormally high antibody levels under resting conditions and show enhanced humoral immune responses after immunization with protein and carbohydrate antigens. Removal of the spleen does not abolish the augmented basal and post-immunization antibody levels in Clever-1-deficient mice. The increased IgG production is also present in mice in which Clever-1 is selectively ablated from macrophages. When compared to wildtype macrophages, Clever-1-deficient macrophages show increased TNF-α synthesis. In co-culture experiments, monocytes/macrophages deficient of Clever-1 support higher IgM production by B cells, which is blocked by TNF-α depletion. Collectively, our data show that the excessive inflammatory activity of monocytes/macrophages in the absence of Clever-1 results in augmented humoral immune responses in vivo.
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Formación de Anticuerpos/inmunología , Linfocitos B/inmunología , Moléculas de Adhesión Celular Neuronal/inmunología , Inmunoglobulina M/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Animales , Linfocitos B/metabolismo , Moléculas de Adhesión Celular Neuronal/deficiencia , Moléculas de Adhesión Celular Neuronal/genética , Técnicas de Cocultivo , Inmunoglobulina M/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Ratones Noqueados , Monocitos/citología , Monocitos/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Current treatment options for castration-resistant prostate cancer (CRPC) are limited. In this study, a high-throughput screen of 4910 drugs and drug-like molecules was performed to identify antiproliferative compounds in androgen ablated prostate cancer cells. The effect of compounds on cell viability was compared in androgen ablated LNCaP prostate cancer cells and in LNCaP cells grown in presence of androgens as well as in two non-malignant prostate epithelial cells (RWPE-1 and EP156T). Validation experiments of cancer specific anti-proliferative compounds indicated pinosylvin methyl ether (PSME) and tanshinone IIA as potent inhibitors of androgen ablated LNCaP cell proliferation. PSME is a stilbene compound with no previously described anti-neoplastic activity whereas tanshinone IIA is currently used in cardiovascular disorders and proposed as a cancer drug. To gain insights into growth inhibitory mechanisms in CRPC, genome-wide gene expression analysis was performed in PSME- and tanshinone IIA-exposed cells. Both compounds altered the expression of genes involved in cell cycle and steroid and cholesterol biosynthesis in androgen ablated LNCaP cells. Decrease in androgen signalling was confirmed by reduced expression of androgen receptor and prostate specific antigen in PSME- or tanshinone IIA-exposed cells. Taken together, this systematic screen identified a novel anti-proliferative agent, PSME, for CRPC. Moreover, our screen confirmed tanshinone IIA as well as several other compounds as potential prostate cancer growth inhibitors also in androgen ablated prostate cancer cells. These results provide valuable starting points for preclinical and clinical studies for CRPC treatment.
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The RNA-binding protein L1TD1 is one of the most specific and abundant proteins in pluripotent stem cells and is essential for the maintenance of pluripotency in human cells. Here, we identify the protein interaction network of L1TD1 in human embryonic stem cells (hESCs) and provide insights into the interactome network constructed in human pluripotent cells. Our data reveal that L1TD1 has an important role in RNA splicing, translation, protein traffic, and degradation. L1TD1 interacts with multiple stem-cell-specific proteins, many of which are still uncharacterized in the context of development. Further, we show that L1TD1 is a part of the pluripotency interactome network of OCT4, SOX2, and NANOG, bridging nuclear and cytoplasmic regulation and highlighting the importance of RNA biology in pluripotency.
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Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Mapeo de Interacción de Proteínas , Proteínas/metabolismo , Procesamiento Postranscripcional del ARN , Secuencia de Aminoácidos , Proteínas Portadoras/química , Proteínas Portadoras/genética , Núcleo Celular/metabolismo , Autorrenovación de las Células/efectos de los fármacos , Autorrenovación de las Células/genética , Citoplasma/metabolismo , Humanos , Datos de Secuencia Molecular , Células Madre Pluripotentes/efectos de los fármacos , Inhibidores de Proteasoma/farmacología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de Proteínas , Transporte de Proteínas , Proteínas/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Early regulators of disease may increase understanding of disease mechanisms and serve as markers for presymptomatic diagnosis and treatment. However, early regulators are difficult to identify because patients generally present after they are symptomatic. We hypothesized that early regulators of T cell-associated diseases could be found by identifying upstream transcription factors (TFs) in T cell differentiation and by prioritizing hub TFs that were enriched for disease-associated polymorphisms. A gene regulatory network (GRN) was constructed by time series profiling of the transcriptomes and methylomes of human CD4(+) T cells during in vitro differentiation into four helper T cell lineages, in combination with sequence-based TF binding predictions. The TFs GATA3, MAF, and MYB were identified as early regulators and validated by ChIP-seq (chromatin immunoprecipitation sequencing) and small interfering RNA knockdowns. Differential mRNA expression of the TFs and their targets in T cell-associated diseases supports their clinical relevance. To directly test if the TFs were altered early in disease, T cells from patients with two T cell-mediated diseases, multiple sclerosis and seasonal allergic rhinitis, were analyzed. Strikingly, the TFs were differentially expressed during asymptomatic stages of both diseases, whereas their targets showed altered expression during symptomatic stages. This analytical strategy to identify early regulators of disease by combining GRNs with genome-wide association studies may be generally applicable for functional and clinical studies of early disease development.
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Linfocitos T CD4-Positivos/inmunología , Redes Reguladoras de Genes , Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunología , Rinitis Alérgica Estacional/genética , Rinitis Alérgica Estacional/inmunología , Linfocitos T CD4-Positivos/metabolismo , Factor de Transcripción GATA3/genética , Estudio de Asociación del Genoma Completo , Humanos , Esclerosis Múltiple/diagnóstico , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas c-maf/genética , Proteínas Proto-Oncogénicas c-myb/genética , Rinitis Alérgica Estacional/diagnóstico , TranscriptomaRESUMEN
Vimentin is an intermediate filament protein, with a key role in the epithelial to mesenchymal transition as well as cell invasion, and it is often upregulated during cancer progression. However, relatively little is known about its regulation in cancer cells. Here, we performed an RNA interference screen followed by protein lysate microarray analysis in bone metastatic MDA-MB-231(SA) breast cancer cells to identify novel regulators of vimentin expression. Out of the 596 genes investigated, three novel vimentin regulators EPHB4, WIPF2 and MTHFD2 were identified. The reduced vimentin expression in response to EPHB4, WIPF2 and MTHFD2 silencing was observed at mRNA and protein levels. Bioinformatic analysis of gene expression data across cancers indicated overexpression of EPHB4 and MTHFD2 in breast cancer and high expression associated with poor clinical characteristics. Analysis of 96 cDNA samples derived from both normal and malignant human tissues suggested putative association with metastatic disease. MTHFD2 knockdown resulted in impaired cell migration and invasion into extracellular matrix as well as decreased the fraction of cells with a high CD44 expression, a marker of cancer stem cells. Furthermore, MTHFD2 expression was induced in response to TGF-ß stimulation in breast cancer cells. Our results show that MTHFD2 is overexpressed in breast cancer, associates with poor clinical characteristics and promotes cellular features connected with metastatic disease, thus implicating MTHFD2 as a potential drug target to block breast cancer cell migration and invasion.
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Aminohidrolasas/genética , Neoplasias de la Mama/genética , Movimiento Celular/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Complejos Multienzimáticos/genética , Interferencia de ARN , Vimentina/genética , Aminohidrolasas/metabolismo , Antineoplásicos/farmacología , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cadherinas/genética , Cadherinas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Proteínas de Microfilamentos , Microscopía Confocal , Complejos Multienzimáticos/metabolismo , Invasividad Neoplásica , Metástasis de la Neoplasia , Receptor EphB4/genética , Receptor EphB4/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/farmacología , Vimentina/metabolismoRESUMEN
Low oxygen tension (hypoxia) contributes critically to pluripotency of human embryonic stem cells (hESCs) by preventing spontaneous differentiation and supporting self-renewal. However, it is not well understood how hESCs respond to reduced oxygen availability and what are the molecular mechanisms maintaining pluripotency in these conditions. In this study we characterized the transcriptional and molecular responses of three hESC lines (H9, HS401 and HS360) on short (2 hours), intermediate (24 hours) and prolonged (7 days) exposure to low oxygen conditions (4% O2). In response to prolonged hypoxia the expression of pluripotency surface marker SSEA-3 was increased. Furthermore, the genome wide gene-expression analysis revealed that a substantial proportion (12%) of all hypoxia-regulated genes in hESCs, were directly linked to the mechanisms controlling pluripotency or differentiation. Moreover, transcription of MYC oncogene was induced in response to continuous hypoxia. At the protein level MYC was stabilized through phosphorylation already in response to a short hypoxic exposure. Total MYC protein levels remained elevated throughout all the time points studied. Further, MYC protein expression in hypoxia was affected by silencing HIF2α, but not HIF1α. Since MYC has a crucial role in regulating pluripotency we propose that induction of sustained MYC expression in hypoxia contributes to activation of transcriptional programs critical for hESC self-renewal and maintenance of enhanced pluripotent state.